Project Summary/Abstract Cell-cell interactions are often mediated by the recognition of N- or O-linked glycans on cell- surface glycoproteins by endogenous lectins. These lectins are often themselves glycoproteins, anchored to cells by trans-membrane segments that generate intra-cellular signals. Glycan mediated interaction of multiple lectin molecules can, therefore, be an important element in regulation of cell to cell signaling. For the past several years this grant has supported the development of nuclear magnetic resonance (NMR) methods for the investigation of structural aspects of the interaction of isolated oligosaccharides with soluble lectins and applied them primarily to a subset of lectins that binds galactose terminated oligosaccharides. We now propose to extend these studies to lectins targeting sialic acid and fucose containing oligosaccharides, and to develop methods that will allow these investigations to proceed when the oligosaccharides are glycans covalently linked to glycoproteins. Modulation of immune response by specific cell-cell interactions is a good example where glycan mediated protein-protein interaction is an issue. B-cell activation is regulated by cis- versus trans- interaction of siglecs (sialic acid binding lectins) on the surface of immune cells. DC-SIGN (dendritic cell -specific ICAM-3 grabbing nonintegrin), a C-type lectin that binds fucosylated oligosaccharides and interacts with ICAM-3 (intercellular adhesion molecule-3) also is believed to modulate an immune response. The proposed studies will provide a structural basis for understanding of these important regulatory mechanisms. The studies will also lead to the development of new methodology that can be applied to a variety of other problems in human biology where glycans mediate protein-protein interactions. The specific aims include expression of siglec domain constructs suitable for NMR studies in glycosylated and non-glycosylated forms, remodeling of glycan residues on siglecs to improve glycan homogeneity and add isotopically labeled sialic acids, development of new NMR resonance assignment strategies for these labeled glycans, collection and interpretation of NMR data to define structure and dynamics of these glycans, and development of parallel strategies that would allow structural characterization of fucose containing glycans important to function of the DC-SIGN system.